This invention provides immunologically active compositions that can induce protective immunity or tolerance. The composition to induce protective immunity both in uninfected or infected host consists of antigenic epitopes but excludes or eliminates epitopes that participate in immune “escape” or induce tolerance. Protective immunity can be induced also by a composition that includes a pathogen associated molecular pattern(s) and/or a carrier with or without the antigenic epitopes. Another immunologically active composition that induces tolerance includes an escape epitope(s) or a molecular pattern(s) important for pathogen escape, with or without a carrier. In addition, the invention provides methods to identify such immunologically active molecules.
Progress in immunobiology has identified the essential immunological factors for the development of immune modulators, including the requirement for inducing innate and adaptive immune responses that control pathogens.
With the advent of widespread antibiotics resistance, immune modulators can be the most effective approach to deliver long-lasting protection against microorganisms including intracellular pathogens. The current focus on immune modulators requires the development of novel vectors, effective carriers and adjuvant systems.
As pathogens can also induce Th2 response and tolerance, this understanding may be used for the development of immune modulators for autoimmune diseases, transplantation and other medical applications. Most pathogens enter the body through the skin and mucosal membranes. Therefore these routes of administration are particularly well suited for immune modulation against infections entering through the skin, the airways, the gastrointestinal tract, or the sexual organs. Traditional vaccines are administered parenterally, far from the actual site of infection and their mucosal response is less pronounced.
It is now apparent that besides Toll-like receptors (TLRs) there are other receptors and pathways that play an important role in the innate immune response. An example for this is the nucleotide-oligomerization domain (NOD) proteins that recognize microbial motifs of intracellular microorganisms. Mindin, an extracellular matrix protein, is also a mediator of inflammatory response to several bacterial surface components. These and other studies suggest that innate immunity involves additional factors independent of TLR signaling and that production of NFκB or IL-1 may not be sufficient to control infections.
Typical elements of innate immunity involved in controlling infections are: (1) proinflammatory response: NF-κB mediated, activates many agents of inflammation, overstimulation can result in shock; (2) cationic host defense peptides: increased production of peptides stimulated by bacterial pathogen associated molecular patterns (PAMPs) and signaling molecules; (3) phagocytic cell activation: increased intracellular killing in neutrophils and macrophages (both oxidative and non-oxidative mechanisms) enhanced, increased cytokine production; (4) chemotaxis: increased endothelial adhesion of phagocytic cells, cell migration to the site of infection, diapedesis; (5) extracellular killing mechanism: complement activation, enhanced iron chelation, antimicrobial peptide secretion, production of degradative enzymes; (6) infection containment: clot formation via fibrinogen activation; (7) wound repair: fibroblast growth and adherence, angiogenesis; and (8) adaptive immune responses: B- and T-cell activation, often via dentritic cells.
Stimulation of innate immunity may be accomplished by using interferons, monophosphoryl-lipid A, imiquimod, CpG nucleotides or cationic peptides. Innate immunity however has a limited capacity to fend off infections and in such scenario the adaptive immune response takes over.
Recently it has been recognized that dendritic cells are essential to link the innate and adaptive immunity and this knowledge allowed immunologists to design immune modulation strategies against poorly immunogenic antigens. Dendritic cells (DCs) originate from precursors of both the myeloid and lymphoid lineages, but are main antigen-presenting cells (APC). DCs are present in every tissue, and during an infection are the key immune cells to enter into contact with the invading pathogen. They are the bridge between the innate and the adaptive immune response.
Endothelial and epithelial cells, monocytes, macrophages and other cells, including immature DCs express pathogen pattern recognition receptors (TLR receptors, lectin domain receptors and other receptors) that bind conserved pathogen associated molecular structures (PAMPs for short) shared by the pathogens such as lipopolysaccharide from Gram negative bacteria, lipoteichoic acid from Gram positive bacteria, peptidoglycan, peptidoglycan-associated lipoproteins, bacterial DNA and flagellin from Gram negative and Gram positive bacteria as well as viral RNA. Different cells express different receptors allowing a tailored response to the pathogen. Upon activation, immature antigen-capturing DC differentiate into mature antigen-presenting DC, able to present antigen in the MHC class-II and class-I contexts, as well as up-regulate the expression of surface co-stimulatory molecules such as CD80 and CD86.
Mature and activated DC migrates to secondary lymphoid organs (lymph nodes, spleen, Peyer's patches), where they translocate to the T-cell areas. The interaction of DC with and stimulation of T-cells is dependent on cytokines, chemokines and adhesion molecules such as intercellular cell adhesion molecules (I-CAMs), leukocyte function associated molecule 1 (LFA-1) and dendritic cell specific ICAM grabbing nonintegrin (DC-SIGN).
Depending on the local cytokine environment and the antigen, cellular T-helper (Th1) and humoral antibody mediated Th2- or Treg-oriented immune responses are triggered to various degrees. The dose of antigen has been shown to direct the Th1/Th2 differentiation, with high doses stimulating preferentially the Th-1 response and low doses the Th-2 response. Carrier devices displaying antigenic proteins and DNA vaccines have been shown to be taken up by immature dendritic cells and lead to an immune response. DC therefore represents a main but not the only target of development for the modulation of the immune system.
Mucosal DCs specifically provide an important first-line of defense by ingesting foreign invaders via both pinocytosis and receptor-mediated endocytosis. DC plays a critical role in mucosal immunity as bodily mucosa act like a barrier between the inside and the outside of the body. DC can be found on the lining of the respiratory tract and of the gut. Langerhans' cells are a population of DC found in skin and mucosa. DCs and M cells transport antigens to the underlying lymphoid follicle that is the immune-inductive site of the gut. Similar nasal and bronchus associated lymphoid tissues have been described in the respiratory tract. This system is important in the gastrointestinal tract, but in the airways, the underlying DC network may be even more important. In the case of oral administration, the immune modulator must pass undegraded through the stomach and the upper intestines. Such degradation is unlikely to occur through a nasal, ocular or genital route of administration. Subsequently, the immune modulator must be taken up through the intestinal epithelium, so it can be adsorbed and subsequently presented to the immune-competent cells by the antigen-presenting cells. The immune competent cells are located in the epithelium, the lamina propria or beneath the basal membrane. Therefore, the immune modulator components must be formulated with a carrier taking them through this barrier. When bound to particulate carriers, it is generally accepted that the molecules can be transported over the barrier by the M-cells in the Peyer's patches.
Premature breakdown or release of the bioactive molecules has hampered the development of particle-based vaccine and drug delivery technologies. This is the likely explanation why in the published literature a high dose of antigen/drug is still required to achieve comparable responses to the injected counterpart. Besides the poor utilization of antigens and drugs, the other main criticism refers to the poor capacity of M-cells in the Peyer's patches (PP) to transport particles and the insufficient immune and other responses induced in humans. The epithelial M-cells of the PP are known to allow the transport of certain bacteria, viruses and protozoa from the intestines. Several studies have shown that a size-dependent uptake with a maximal diameter of 10 μm may occur by M-cells, DCs and Caco-2 cells.
Recent information on the uptake of particles by M-cells and the different types of dendritic cells (DCs) present in the PPs and their vicinity may provide an understanding of the mechanisms involved. Beyer (Beyer T., et al; Bacterial carriers and virus-like-particles as antigen delivery devices: Role of dendritic cells in antigen presentation. Curr. Drug Targets-Infect. Disord, 2001 1, 287-302) followed the uptake and kinetics of Baker's yeast cells (Saccharomyces cerevisiae) into PP, assuming it as an inert model for transport through the mucosa. A typical time dependency compatible with the transport to different types of phagocytosing, antigen-processing macrophages or dendritic cells was found for the distribution of the yeast cells in the M-cells, the intercellular pocket below the M-cells and the space beneath the basal membrane. Depending on where the DCs are located, they were found to have different functions in the PP microenvironment producing Th1 or Th2-directing cytokines upon activation. The cytokine and chemokine microenvironment will then subsequently decide the differentiation of the Th-cells to Th1 and Th2 subsets, respectively and affect the survival or apoptosis of T-cells, as well.
In addition, the differentiation of the B-cells and the homing of the mucosal plasma cells are regulated by separate cytokines (IL-6 in addition to TGF-β, IL-4, IL-5, and IL-10) and the specific homing receptor, a4b7. It can thus be concluded that there seem to be mechanisms available in the mucosa, by which mucosal modulation of the immune system can induce a more differentiated immune response, better mimicking the response to a natural infection than obtained by other routes of administration.
Although much has been learned about DC, their precursors and various DC subtypes that have been proposed, the full degree of functional complexity and plasticity of DC renders difficult predictions about the effect of a specific vaccine on DCs and subsequent Th1, Th2 and Treg responses. However, some of the results obtained for DC matured in vitro might be extrapolated to mature DC isolated from the lymphoid organs since they display similar characteristics (Shortman K., et al; Mouse and human dentritic cell subtypes. Nat. Rev. Immunol. 2002 Mar. 2(3): 151-61). For instance, the potential of the mycoplasma lipopeptide MALP-2 to modulate DC response has been studied in vitro (Weigt H., et al; Synthetic mycoplasma-derived lipopeptide MALP-2 induces maturation and function of dendritic cells. Immunobiology 2003, 207(3): 223-33). MALP-2 treatment of DC induced the expression of CD80, CD86 and the release of bioactive TNF-α and IL-10, as well as the proliferation of autologous lymphocytes and the production of IL-4, IL-5 and γ-INF by the latter. These features correlate with an ability to stimulate T-cells and therefore suggest a possible effect of MALP-2 on DC in vivo.
Synthetic carriers may enable the immunostimulating effect of antigens for MHC Class I and II presentation. Synthetic carriers may be developed into a versatile system that can be tailored to a variety of potential applications. The character of these carriers can significantly influence the outcome and efficiency of the immune response. Synthetic carriers, such as particles may ease the hurdles of quality assurance and validation in vaccine development and production, and thus shorten the time for approval and to the market.
Several immune stimulatory components (peptides, proteins, lipids or polysaccharides) of different infectious microorganisms can be used for vaccination. These components can be synthesized, purified from the microorganisms or produced by recombinant DNA technology. However, they require suitable adjuvants when administered in free, soluble form orally or parenterally.
[Several particle-based systems have been tested as carriers for various antigens and drugs. Chitosan, poly-DL-lactic acid, or polyacryl starch micro particles have previously been described as a drug carrier system. Examples of such systems are described in U.S. Pat. Nos. 5,603,960 and 6,521,431. In one report, it was observed that starch micro-particles with covalently bound human serum albumin (HSA), as a model antigen functioned as a strong adjuvant in mice when administered parenterally and the micro particles alone were not immunogenic.
It should be pointed out, though, that the uptake is most likely dependent on the structure and the possible adhesive properties of the carrier, too. Agarose and other polysaccharides have intrinsic mucoadhesive properties, which may improve their interaction with different mucosal membranes and facilitate uptake.